The goal of this project is to define the molecular mechanisms involved in the replication of HIV and related retroviruses and to develop new strategies for AIDS therapy. Our research is currently focused on two broad areas of interest: reverse transcription and virus assembly. During reverse transcription, there are two strand transfer events that are required for synthesis of full-length plus- and minus-strand DNA copies of the viral RNA genome. This process is dependent on the viral nucleocapsid protein (NC), a nucleic acid chaperone with the ability to catalyze conformational rearrangements that lead to the most thermodynamically stable nucleic acid structures. HIV-1 NC has two zinc fingers, each containing the invariant CCHC zinc-coordinating motifs; however, other residues within each finger are not identical. (A) A question of major interest concerns the functional significance of the two zinc fingers in HIV-1 NC. Using a mutational approach, we have provided the first direct evidence that zinc coordination by the CCHC motifs (and not by alternative zinc-binding motifs) as well as the correct amino acid context surrounding the CCHC motifs are required for efficient strand transfer. The zinc fingers are also required for NC-mediated inhibition of dead-end self-priming reactions, which compete with minus-strand transfer and are induced by the complementary TAR secondary structure at the 3-prime end of (-) strong-stop DNA. To gain a better understanding of how NC blocks self-priming, our collaborator Karin Musier-Forsyth and colleagues have developed a fluorescence resonance energy transfer (FRET) assay, that makes it possible to directly monitor conformational changes in TAR DNA when NC is present. When NC binds to TAR DNA alone, there is only a modest shift towards less-folded states. However, in the presence of acceptor RNA, NC binding to TAR DNA results in a shift of the majority of molecules to the unfolded state. These data are completely consistent with biochemical data obtained under similar conditions. Work is now in progress to define the structural requirements for NC interaction with strand transfer nucleic acid intermediates. (B) Other studies are being performed on the initiation step in HIV-1 reverse transcription. This event is primed by a host lysyl-tRNA, which is annealed to the 18-nt primer binding site (PBS) near the 5-prime terminus of the viral RNA genome; extension of the primer leads to synthesis of (-) strong-stop DNA. To investigate a possible role for sequences downstream of the PBS in this step, we assayed RNA templates with varying lengths of downstream sequences, using three different primers: lysyl-tRNA; and 18-nt RNA (R18) or DNA (D18) oligonucleotide mimics, having the sequence of the 3-prime end of the tRNA. Interestingly, a minimum of 24 bases immediately downstream of the PBS is required when the RNA primers, but not the D18 primer, are used. Correlation of the priming activities of R18, D18, and 18-nt chimeric DNA-RNA primers with circular dichroism spectra and melting studies of PBS:primer duplexes point to the importance of helical conformation and stability as determinants of priming activity. Surprisingly, when NC is present, the additional downstream bases are dispensable for synthesis primed by the tRNA, but not for synthesis primed by the R18 primer. We propose that NC facilitates stable formation of extended interactions between tRNA and the RNA template, which are not possible with an 18-nt RNA. (C) Relative to HIV-1, studies of HIV-2 infection have been limited. Both viruses share similar genome organization, protein composition, and mode of transmission, but differ in time of onset of AIDS and geographic pattern of infection. In addition, drugs that target HIV-1 are not necessarily effective against HIV-2, posing a major problem for clinical treatment of HIV-2-infected individuals. We have therefore conducted a systematic evaluation of HIV-2 reverse transcriptase (RT) function, a target for antiviral therapy, using assays that model steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes are comparable. In contrast, when the RT concentration is severely reduced, HIV-2 RT activity is considerably lower than that of HIV-1 RT. HIV-2 RT is also impaired in its ability to catalyze secondary RNase H cleavage in an assay that mimics tRNA primer removal during plus-strand transfer. In addition, initiation of plus-strand synthesis is much less efficient with HIV-2 RT compared with HIV-1 RT, unless the salt and magnesium ion concentrations are drastically lowered. This may reflect architectural differences in the Primer Grip regions of the two enzymes. Taken together, our findings should be valuable for development of specific high-throughput screening assays of potential HIV-2 inhibitory agents. Our laboratory has also been investigating the role of the HIV-1 capsid protein (CA) in early postentry events, a stage in the infectious process that is still not completely understood. In a recent report, we described the unusual phenotype associated with single alanine substitution mutations in conserved N-terminal hydrophobic residues. Mutant virions are not infectious and lack a cone-shaped core. Moreover, despite having a functional RT protein, the mutants are blocked in viral DNA synthesis in cells, indicating a defect in an early postentry event preceding reverse transcription. Current efforts have focused on elucidating the mechanism by which these CA mutations disrupt virus infectivity. The new work has uncovered several novel properties of the mutants. We have observed that the mutations block the incorporation of host cyclophilin A (a peptidyl-prolyl cis-trans isomerase required for virus replication) into virions. This finding was unexpected, since the mutated residues are distant from the cyclophilin A binding loop in CA. These results indicate that the mutations induce conformational changes in CA that have global effects on CA structure and function. Additionally, mutant cores have a severely reduced level of RT; they also retain an extraordinarily high amount of CA, indicating that the cores are unusually stable. This would interfere with proper disassembly and taken together with the RT defect, would also account for the failure of the mutants to synthesize viral DNA postentry. Our results reveal for the first time, the crucial role of the N-terminal hydrophobic core of CA in HIV-1 replication. The critical importance of these residues for maintaining CA structure and function indicates that this motif represents a potential new target for antiviral drugs and other therapeutic approaches.